B cells produce antibodies, and each B cell produces an antibody having one particular antigen specificity (i.e. , the antibody is monospecific). If one expands a single B cell clone, the antibodies produced by the expanded population of B cells are homogeneous. Hybridoma technology, which has been in existence since 1975, enables one to immortalize individual B cells, and thereby allows one to expand a population of individual B cell clones to obtain sufficient antibodies so that the immortalized population can be screened to isolate those B cells producing antibodies having a particular antigen specificity. The B cell hybrids of interest can be grown in a large scale to make large quantities of homogeneous monoclonal antibodies, which are useful for diagnostic and therapeutic purposes. Kennett, R. H., et al, Monoclonal Antibodies (Plenum Press, New York 1980); Borrebaeck, C. A. K. and Larrick, J. W., Therapeutic Monoclonal Antibodies (Stockton Press, New York 1990); Winter, G. and Milstein, C. Nature 349:293 (1991).
Hybridoma technology generally works best for preparing purely murine monoclonal antibodies. Hybrids made from fusing human B cells with human or murine myeloma cells are generally unstable, and tend to lose the human chromosomes and the ability to produce antibodies. Transforming human B cells with the Epstein-Barr virus (EBV) offers an alternative way to immortalize them. However, the transformation frequency is relatively low and the transformants are also not stable and often lose antibody-producing ability.
An alternative method, often referred to as the "combinational V region library" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity. Sastry, L. et al., Proc. Natl. Acad, Sci. U.S.A. 86:5728 (1989); Huse, W. D. et al., Science 246:1275 (1989); Orlandi, R. et al., Proc. Natl. Acad. Sci. U.S.A. 86:3833 (1989). This technique relies on polymerase chain reaction (PCR) techniques. Using degenerate oligonucleotide 5'-end primers corresponding to the leader peptides or the N-terminal framework segments of V.sub.H, V.sub..kappa., and V.sub..lambda., and 3'-end primers corresponding to the CH1 domain of a heavy chain such as .gamma. and to the constant regions of light chains .kappa. and .lambda., the V.sub.H, V.sub.78 , and V.sub.80, regions of a population of B cells are amplified with PCR. The pools of these V.sub.H, V.sub.78, and V.sub.80, genes are referred to as V.sub.H, V.sub..kappa., and V.sub. 80, libraries of a particular B cell population. Using specially designed vectors derived from a bacteriophage, a V.sub.H segment and a V.sub.L segment can be inserted into the vector and coexpressed in an E. coli bacterial host cell. The random combination of the V.sub.H and V.sub.L libraries creates an extremely large VI.sub.H -VI.sub.L combinatorial library. Each pair of combined V.sub.H and V.sub.L forms a unique antibody Fv fragment with a particular antigen-binding specificity.
The recombinant .lambda. phage including the expression vectors that contain the combined V.sub.H and V.sub.L genes can be cloned, and then various methods can be applied to determine the antigen-reactivity of the antibodies which are expressed. In one earlier expression system, the combined V.sub.H and V.sub.L gene segments in bacteriophage .lambda. were expressed as soluble Fab fragments in E. coli. In a more recently developed system, the combined V.sub.H and V.sub.L fragments are expressed as a part of a protein on the surface of a filamentous bacteriophage, designated fd. In this latter system, the bacteriophage expressing the antibody fragment of interest can be isolated using affinity chromatography. McCafferty, J. Nature 348:552 (1990).
When using EBV transformation or cell fusion as described above to obtain monoclonal antibodies of particular specificity, a number of factors determine the probability of isolating the desired antibodies. Some of these factors are:
1. The pre-fusion number of B cells expressing the desired antibodies in the B cell population (referred to as the frequency of desired B cells); PA0 2. The number of B cells in the B cell population which are fused with myelomas or transformed by EBV (respectively referred to as the frequency of fusion or transformation);
3. The stability of the immortalized cells.
Referring to factor "1" above, the frequency of desired B cells in mice is affected by whether the mice from which the B cells are derived are unexposed, immunized, or hyperimmunized with the antigen. By some estimates, the antibody repertoire in a mouse could represent over 10.sup.10 different antibody species. However, at any given time, there may be only 10.sup.7 -10.sup.8 B cell clones in a particular mouse. Winter, G. and Milstein, C. Nature 349:293 (1991). This suggests that for most antigenic epitopes, the frequency of B cells producing reactive antibodies may be in the order of 1.times.10.sup.-5 or 1.times.10.sup.6, or less. It is also known that in a hyperimmunized mouse, the frequency of B cells specific for the immunogen may account for several percent of the total, or more.
When making human monoclonal antibodies, the frequency of desired B cells is affected by whether the human donors from whom the B cells are derived are immunized with the antigen through infection, vaccination, or other exposure. Depending on the intensity of a viral infection, the stage in the immune response, and the immunogenicity of the particular viral antigen, the frequency of B cells in the infected person specific for a particular antigenic epitope on a viral protein may range from 1.times.10.sup.-3 to 1.times.10.sup.-5 for the more dominant epitopes and less than 1.times.10.sup.-5 for the relatively silent epitopes. The frequency of IgG-expressing B cells specific for a protein allergen will often vary among different individuals, and may vary with the concentration of the allergen in the environment. The frequencies of B cells specific for autologous antigens, such as IgG, platelet surface antigens, leukocyte surface antigens, nuclear proteins, or DNA, will also vary among different individuals. For a relatively nonimmunogenic antigenic epitope on a large protein molecule, such as the CD4 binding site on gp120 of HIV-1, the B cells expressing antibodies specific for an antigenic epitope thereof in a moderately immunized mouse or an HIV-1 infected person may only occur at a frequency of 1.times.10.sup.-5 or lower among the IgG-bearing B cells. For an antigen the B cell donor has not been exposed to, the frequency of B cells specific for the antigen may be 1.times.10.sup.6 or lower.
In a fusion procedure for preparing murine hybridomas, immunized mouse spleen cells are fused with myeloma cells. The number of lymphocytes in a mouse spleen is about 1.times.10.sup.8. B cells account for about 10% of the total, and among B cells, IgG-expressing B cells account for about 20%. Thus, in a mouse spleen, there are about 2.times.10.sup.6 IgG-expressing B cells.
In performing a cell fusion or EBV transformation procedure to immortalize IgG-expressing B cells from a person, one may typically take 100 ml peripheral blood from the donor. There are about 1.times.10.sup.8 lymphocytes in this amount of blood. Among them, B cells account for about 1-1.5.times.10.sup.7, and IgG-expressing B cells account for about 2-3.times.10.sup.6.
If the targets of immortalization are antigen-specific IgG-expressing B cells occurring at moderately low frequency, for example, 1.times.10.sup.-5 among the IgG-bearing B cells, there will only be about 20-30 such B cells in a mouse spleen or in 100 ml of peripheral human blood.
The difficulty which stems from having such a small number of B cells producing the desired antibody relates to factor "2" above: the frequency of cell fusion or transformation. A skillful technician using a good fusion protocol can generate about 10,000 hybrids from the approximately 1.times.10.sup.7 B cells which are taken from the spleen of a mouse. This corresponds to a fusion frequency of 1 in 1000 B cells. The cell fusion and EBV-transformation frequencies for human B cells are at least about 1 to 2 orders of magnitude lower. Thus, if one is starting with only about 20-30 B cells with the desired antigenic specificity, the chances of immortalizing one of the desired B cells is very slim.
Factor "3" above refers to the stability of the immortalized cells. As noted above, while murine hybridomas are generally stable, human hybridomas and EBV transformants are much less stable. Sequential subcloning is usually required to obtain stable human hybrids and transformants. However, even with such subcloning, which can be a laborious procedure, stable antibody-secreting cell lines cannot be obtained for a large portion of human hybrids and transformants.
While the combinatorial library methodology can be used to produce antigen-specific antibody fragments in bacterial host cells, it also suffers certain drawbacks related to the low frequency of antigen-specific B cells. For determining whether a particular B cell expresses antibody molecules of desired antigen-binding specificity, the V.sub.H and V.sub.L segments of the mRNAs of the heavy and light chains need to be reverse transcribed into cDNA, amplified by PCR, and the resulting gene segments must be cloned, sequenced, and incorporated into expression vectors. The two gene segments must then be coexpressed in a mammalian cell or other system, and the fragments produced must be characterized for antigen-binding specificity and for relative binding affinity. In a natural immune response, the B cells expressing the antibodies specific for an antigen expand, resulting in higher representations of such cells among the B cell repertoire. The combination of the desired V.sub.H and V.sub.L gene segments are expressed only by the antigen specific B cells. The combinatorial library methodology separates the V.sub.H and V.sub.L genes and recombines them in a random fashion. If B cells secreting an antibody of desired antigen-binding specificity are present at a frequency of 1.times.10.sup.-5, the combinatorial library will expand the V.sub.H -V.sub.L library. Depending on the diversity of the repertoire in the V.sub.H and V.sub.L libraries, the original V.sub.H and V.sub.L combination will be present at a frequency of probably about 1.times.10.sup.-10. It has been suggested that certain V.sub.H -V.sub.L combinations resulting from this genetic manipulation may also have the desired antigen specificity. However, this possibility is yet to be substantiated by experimentation. Winter, G. & Milstein, C. Nature 349:293 (1991).
Screening large numbers of bacteriophage particles expressing the desired fragment is generally more efficient than screening hybridomas or EBV transformants for the corresponding antibody. The typical screening methods for bacteriophage plaques can handle up to 10.sup.6 or 10.sup.7 phages, assuming that the availability of antigen suitable for the antigen-binding assays is not a limiting factor. However, for certain soluble antigens or antigenic epitopes, screening bacteriophages incorporating the reactive antibodies is difficult, because the antigens are limited or only available in soluble form. Additionally, for certain other antigens including membrane-bound proteins, the conformational epitopes on the antigens are altered if the antigens are solubilized from the membrane, and they will not react with the fragments being screened.
Additionally, if one is screening the antibody-expressing bacteriophages on an immunoblotting plate, about 50,000 plaques can be screened on one plate. In an extremely ambitious experiment, 100 plates may be screened. This screens about 5.times.10.sup.6 plaques or phages, which is much below the theoretical number of plaques which need to be screened to obtain the desired fragment.
A new method has been described for expressing the antibody fragment V.sub.H -V.sub.L as a part of a protein on the surface of filamentous phage. It has been claimed that the phage expressing the specific antibody can be affinity purified from a large number of phages using an antigen-conjugated affinity column. McCafferty, J. et al., Nature 348:552 (1990). Theoretically this sounds plausible, however, it is yet to be proven that the methodology can identify and purify one specific phage from 1.times.10.sup.10 phages. In addition, one major problem in immunoblotting and affinity chromatography methods is that antibodies with a moderate affinity for the antigen will be selected. This allows the inclusion of many cross-reactive or sticky antibodies, causing burdens in the sequential screening procedures.
Various methods have been employed to enrich the desired B cells when they occur at low frequency. These methods can be used to enrich the desired B cells whether one is attempting to isolate the V.sub., and V.sub.L fragments by hybridoma fusion technology, EBV transformation, or the combinatorial library methodology. These enrichment methods include fluorescence-activated cell sorting (FACS), panning against antigen-coated plastic surface, binding to antigen-coated magnetic beads, or resetting with antigen-coated red blood cells.
These procedures for enriching antigen-specific B cells, however, all suffer from a certain degree of nonspecificity. For example, panning or absorbing to plates or beads yields from about one to several percent of nonspecific binding. Rosetting of cells by antigen-coated red blood cells also has about the same degree of nonspecific activity. For cell sorting using FACS, the nonspecific sorting will depend on the stringency of the gate setting, but it usually ranges from 0.1 to several percent depending on the nature of the antigen.
An additional problem in a typical FACS procedure where mixed leukocytes are screened is background noise. An FACS apparatus operates by having a number of channels, which can each view a different fluorochrome. Single cells can be selected based on their fluorescence, or lack of fluorescence. Background noise in an FACS apparatus arises primarily from two sources. One source of noise is exhibited by certain activated cells or proliferating cells of various leukocyte subpopulations that contain high concentrations of certain metabolites, which cause these cells to exhibit autofluorescence even in the absence of fluorochrome labeling. Another possible source of background noise arises because some of the activated cells, and some monocytes and macrophages, possess sticky cellular plasmamembranes. Fluorescent probes will non-specifically adhere to the sticky plasmamembrane and create an additional source of background fluorescence in the cell sample.
Assuming that the B cells expressing antibodies having the desired antigen-binding properties in a human donor occur at a frequency of about 10.sup.-5 to 10.sup.-6 (which is a reasonable estimate for B cells specific for a weak antigenic epitope in an individual with exposure to the antigen or an individual naive to the antigen) the B cell enrichment procedures discussed above are not useful. If an enrichment method provides 1% nonspecific activity, then the desired B cells will average only about one in 1000 or 10,000 B cells isolated. Obviously, the single cell-PCR procedure to be described below for identifying and isolating antibodies of desired antigen-binding specificity would not work at such a low frequency. Thus, a method to increase the proportion of antigen-specific B cells among the single B cells selected for performing the single cell procedure is needed.